Many academic and industrial processes utilise cell culture to generate cells or in the production of biomaterials or compounds of interest.
In other processes, such as ultra filtration or the mixing of biological solutions or the dissolution of solids into aqueous solutions, it is necessary to provide fast, effective and yet gentle mixing of the liquid medium.
In cell culture, an aliquot of cells is placed in a vessel of some description, provided with the nutrients required for the growth of the cells, and is either supplied with oxygen or grown anaerobically in the absence of oxygen. After a period of time to allow production and growth of new cells, the cultured cells are typically removed from a vessel and harvested or separated from the medium.
In processes upstream and/or downstream from the culture process, it is often important to mix cells, media or other materials, to prevent them from settling in a storage tank or from forming precipitates. In these instances, it is essential to keep the medium contained within the vessel from settling and so it is advantageous for the medium in the vessel to be kept in constant motion.
Since the first use of aerobic cell culture systems, there have been a series of physical constraints that workers in the field have attempted to address. One of the primary constraints is how to achieve good aeration of the culture without incurring (excessive) damage to the cells being grown and at the same time to achieve a homogenous suspension, or mixing, of cells and to avoid the formation of unstirred areas or “dead spots”.
One arrangement widely used in cell culture employs sparging air in the bottom of a vessel so as to cause recirculation of the culture medium. This further improves aeration without the need for physical agitation and has the added benefit of mixing the cell culture without the need for expensive stirring, rolling or rocking devices. The type of culture system that uses an internal air-powered aeration system is often described as a pressure-cycle fermenter or airlift fermenter.
In a pressure-cycle fermenter, the aerated stream of gas and solution that results from sparging the vessel at its base, has a lower density than that of the undisturbed solution and therefore rises. This stream is often referred to as the “riser”. The solution that is drawn in to take its place forms a stream, often referred to the “downcomer”. Circulation within the cell culture can therefore be achieved simply by aerating the culture as described in GB2002417A, U.S. Pat. No. 5,660,977 and GB 1383432A.
However this method often suffers from having regions of ‘dead zones’ in which the culture media is not effectively mixed. To overcome this problem, many inventions have sought to separate the riser and the downcomer through the use of a physical barrier. GB2002417A, U.S. Pat. No. 5,660,977 and GB1383432A describe a variety of configurations in which this is achieved. Generally, the riser is directed to the surface of the culture solution through a tube that is arranged vertically and runs from the bottom, up to just below the surface of the culture medium. The riser moves upwards, and is replaced by the downcomer which circulates the solution. In other arrangements, the downcomer can be an external tube.
Other arrangements. such as that disclosed in U.S. Pat. No. 4,649,117, do not require the use of a separation device to divide the riser and the downcomer. In this arrangement, the upper surface of the culture medium is considerably wider than at the base, which contains the sparger. While this will generate the required riser and downcomer, it can be observed in such systems that there are often areas of the culture that are mixed less well than others to give “dead spots” or “flat spots”. To compensate for this, the operator will often increase the aeration rate to achieve better mixing. This is a disadvantage because increased sparging will also give rise to the requirement for more gas, which increases process cost, and greater foaming. Foaming must be controlled, as for certain mammalian cell culture systems, there is a direct correlation between increased sparge rate and increased cell mortality. This trend can be entirely eliminated by controlling foaming through the addition of antifoaming agents. One skilled in the art will recognize that a better solution to this problem is to keep foaming to a minimum so as to reduce the cost of the antifoam addition or the possible interference of the antifoam reagents in downstream processes.
JP61202680A teaches that cells attached to a carrier (generally a bead with an activated surface to which cells adhere) can be kept in suspension in a chamber that is separated from the media feed and mixing apparatus by a porous plate that allows the passage of media and gas but does not allow the carriers, with their adherent cells, from passing through the porous separator plate. This is an advantage because the beads are kept separate from the stirring means, which tend to fragment the beads and therefore loose their ability to bind cells when damaged. However, the porous plate cannot separate cells that are grown in free suspension, which would therefore pass through the porous plate. Therefore JP61202680A is limited in the type of cells which can be successfully grown, and prevent it from being applied in the growth of cells such as insect cells, yeast and bacteria, all of which are grown in suspension and not on surfaces.
The costs of assembling and maintaining such fermentation and cell culture systems in a production environment has always been high and steps have therefore been taken to develop disposable systems made from low-cost plastics. EP 0 343 885 discloses a system that utilises the pressure-cycle principle in a plastics bag, in which the riser and downcomer are separated by an intervening plastics sheet, to promote the pressure-cycle effect. The plastics vessel is sparged using a tube inserted from the top of the plastics vessel and the air is dispersed into small air bubbles through the use of a metallic frit assembly. While the use of a separating sheet may in theory provide better circulation due to the pressure-cycle process, overall, it reduces the amount of mixing that takes place and inevitably causes “dead zones” as mentioned above. A very similar arrangement of a reusable plastics bag with a downward-pointing sparging pipe was described in US 2002/0110915 A1 in which no separator was employed.
Many of the commercially available cell culture systems have relied on the use of some form of impeller, under which the aerating gas is delivered into the cell culture. The speed at which the impeller is rotated, its shape and surface area all have the potential to improve the rate of oxygen transfer. A good summary of the art of impeller design can be found in U.S. Pat. No. 6,250,797. One of the major disadvantages of agitation with impeller blades of any shape is that they cause shear stress and often lead to excessive foaming of the cell culture. Considerable heat can also be generated which must be removed, further increasing costs for production. These issues are to be avoided with non-bacterial cells as this results in lower biomass production and poor cell viability.
Other methods of improving oxygen transfer rates without inducing shear stress as a result of deploying impellers have been developed. For example, flexible plastics bags provide a number of opportunities for alternative means of gentle agitation. Bags can be manipulated through the use of external pressure applied mechanically (U.S. Pat. No. 3,819,158), by moving liquid from one bag chamber to another (EP 972 506 A), from rocking the bag back-and-forth (U.S. Pat. No. 6,190,913) or by moving one portion of the bag using mechanical means with pneumatic collars or cuffs (US 2005/0063250 A). In these cases, it is still often necessary to enrich the airflow into the air space above the culture in order to achieve sufficient rates of oxygen transfer and such systems are only used to grow cells with a low requirement for oxygen.
These issues are not confined to cell culture, but also apply in the preparation of solutions and solids that are used in bio production of all types, or in the isolation of the bio product from the cell culture medium. The use of a disposable modules in such operations significantly reduces the cost of cleaning and revalidation of the plant's components compared with modules made from glass and stainless steel. However, the provision of effective mixing has remained a major challenge for many of the reasons already discussed. Agitation must be achieved either by recalculating a liquid at great velocity, which itself causes problems, or through the inclusion of a disposable propeller driven directly through a sealed bearing system (which must be disposable) or through the use of a magnetically coupled and disposable propeller. Both generate heat, excessive shear forces and are expensive.